Multicellular Tumor Spheroids for Evaluation of Cytotoxicity and Tumor Growth Inhibitory Effects of Nanomedicines In Vitro: A Comparison of Docetaxel-Loaded Block Copolymer Micelles and Taxotere®

Abstract

While 3-D tissue models have received increasing attention over the past several decades in the development of traditional anti-cancer therapies, their potential application for the evaluation of advanced drug delivery systems such as nanomedicines has been largely overlooked. In particular, new insight into drug resistance associated with the 3-D tumor microenvironment has called into question the validity of 2-D models for prediction of in vivo anti-tumor activity. In this work, a series of complementary assays was established for evaluating the in vitro efficacy of docetaxel (DTX) -loaded block copolymer micelles (BCM+DTX) and Taxotere® in 3-D multicellular tumor spheroid (MCTS) cultures. Spheroids were found to be significantly more resistant to treatment than monolayer cultures in a cell line dependent manner. Limitations in treatment efficacy were attributed to mechanisms of resistance associated with properties of the spheroid microenvironment. DTX-loaded micelles demonstrated greater therapeutic effect in both monolayer and spheroid cultures in comparison to Taxotere®. Overall, this work demonstrates the use of spheroids as a viable platform for the evaluation of nanomedicines in conditions which more closely reflect the in vivo tumor microenvironment relative to traditional monolayer cultures. By adaptation of traditional cell-based assays, spheroids have the potential to serve as intermediaries between traditional in vitro and in vivo models for high-throughput assessment of therapeutic candidates.

Spheroids Formation on Non‐Adhesive Surfaces by Liquid Overlay Technique: Considerations and Practical Approaches

“…Cisplatin Breast cancer EMT-6 [24] Endometrial cancer Ishikawa [25] RL95-2 [25] KLE [25] Hypopharyngeal cancer FaDu [96] Ovarian cancer OVCAR8 [97] Oral cancer SCC25 [96] CAL27 [96] Chlorimipramine Glioma C6 [98] Cyclophosphamide Breast cancer EMT-6 [24] Diclofenac Liver tissue Hepatocytes obtained from patient [10] Hepatoma C3A [68] Docetaxel Cervical cancer HeLa [99] Colon cancer HT29 [99] Doxorubicin Cervical cancer HeLa [70] Endometrial cancer Ishikawa [25] RL95-2 [25] KLE [25] Hepatoma C3A [68] Huh7 [100] Lung cancer A549 [101] E-combretastatin A4 Melanoma C8161 [102] Breast cancer MDA 231 [102] (Continued) [26] PC3 [26] Fialuridine Liver tissue Hepatocytes obtained from patient [10] Fialuridine Hepatoma C3A [68] Gant61…”

Drug delivery to solid tumors: the predictive value of the multicellular tumor spheroid model for nanomedicine screening

“…After spheroid formation (Table 1) and incubation with the drug for various periods of time, measurements of spheroid growth kinetics (ie, spheroid integrity, growth inhibition and regrowth after treatment) are performed. 43 They are followed …”

“…by the determination of cell viability (eg, acid phosphatase or lactate dehydrogenase release assays) 43,44 and/or cell survival by testing the ability of a single cell to grow into a colony by clonogenic assay. 40,45 Overall, MCTS is more resistant to the treatment than 2D cell culture and can recapitulate the drug resistance, observed in solid tumors.…”

“…59,69 Estimation of drug concentration in MCTS could be done by high-performance liquid chromatography (HPLC), 20 flow cytometry or chemical extraction with successive fluorescence measurements. 43,44,57,64 After incubation, MCTS is collected and embedded in special medium as tissue-Tek before being cut in thin sections (~5 µm).…”

“…MCTS with lower cell density and larger extracellular space enable NP migration, whereas stroma-embedded MCTS turns out to be an appropriate model to demonstrate the barrier effect of stromal components on NP penetration. 34,43 The second main factor is a design of NPs that takes into account various parameters such as size, 20,[60][61][62] composition, 72 surface charge and coating of the nanocarriers. 20,64 These properties are valuable indications for the optimal design of nanocarriers, allowing improved penetration in MCTS and treatment effectiveness.…”